﻿The biosynthesis, regulation, and transport of methanobactins

Grace Eileen Kenney

doi:https://doi.org/10.21985/N22F44

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The biosynthesis, regulation, and transport of methanobactins

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Methanobactins (Mbns) are a growing family of ribosomally produced, post-translationally modified natural products that exhibit a high affinity for copper. These compounds were initially identified in methanotrophic bacteria, which oxidize methane to methanol as their sole source of carbon. One of the key enzymes in this metabolic pathway, particulate methane monooxygenase, is a copper enzyme, and methanotrophs have a consequentially high need for copper. Under low-copper conditions, Mbns are synthesized from ribosomally produced peptides, post-translationally modified, and secreted from the cell, where these compounds can liberate otherwise bioinavailable copper from mineral and organic sources. The characteristic post-translational modifications of Mbn, namely two paired nitrogen-containing heterocycles and neighboring thioamide groups, are responsible for Mbn’s notably high copper binding affinity. After binding copper, CuMbn is taken back up into the cell, where the copper enters the cellular copper pool. In 2010, when the work described in this dissertation began, the extant literature on Mbns was limited to the structure, metal binding affinity, and extracellular localization of the compound produced by Methylosinus trichosporium OB3b. There has been significant progress in understanding Mbns and their role(s) in copper homeostasis in the years since, and the research described in this dissertation represents a substantive component of that progress. Initial efforts were successfully directed towards proving that intact copper-loaded Mbns are actively reinternalized by methanotroph cells, but identification of the putative importer proved challenging. After the Ms. trichosporium OB3b genome was sequenced, this changed. Among the many genes encoding hypothetical proteins was a gene encoding a small peptide with C-terminal similarity to the peptidic Mbn backbone. Another group identified this gene, which prompted the next stage of this project: a genome mining study in which 18 Mbn operons from 16 bacterial species were identified. The components of these operons were analyzed to gain insight into the mechanisms of Mbn biosynthesis, regulation, and transport. In the intervening years, the number of Mbn operons has increased to 74 from 71 bacterial species, but the conclusions from this work have remained applicable to these new Mbns. To provide biological support for the data from the genome mining project, a set of qRT-PCR studies were designed to monitor the time-dependent changes in gene expression of copper starved Ms. trichosporium OB3b cells in response to the addition of copper. These experiments confirmed that the Mbn operon is down regulated in the presence of surplus copper, as is to be expected for a natural product involved in copper acquisition. A related set of studies in a constitutively Mbn-producing strain helped to identify other genes with a potential role in copper homeostasis in this methanotrophic organism. Confirmation of the copper-dependent regulation of the Mbn operon provided vital evidence that the operon had indeed been correctly identified. This justified the experimental exploration of hypotheses regarding the roles of proteins encoded by the operon. A wide range of strategies were utilized: experiments involving the secretion or uptake of Mbn in wild-type or mutant methanotroph strains provided important in vivo evidence for the function of several proteins related to transport and biosynthesis, particularly within the model organism Ms. trichosporium OB3b. However, these experiments could shed no light on the mechanisms behind any of the biochemical or biophysical interactions. To fill that gap, heterologous expression and in vitro experiments that probed substrate binding affinity and potential reactivity provided the first direct biochemical evidence for the roles of several key proteins – including the Mbn importer that had proved so elusive at the beginning of this project. While not every member of the Mbn operon has been probed in vivo and in vitro, a significant subset now have experimental evidence to support the roles that were initially assigned to them via bioinformatic analyses. This dissertation lays the groundwork for several major areas of future research. Mbn is a prototype for natural product-mediated copper influx as part of bacterial copper homeostasis, and research into both Mbns from non-methanotrophs and unrelated natural products that play similar roles will expand our understanding of metal homeostasis in bacteria. The enzymes involved in Mbn biosynthesis mediate novel biochemical reactions, and investigations of their mechanisms will likely prove quite interesting. Finally, as ribosomally produced and post-translationally modified natural products, this class of compounds is well suited to high throughput screening of artificially encoded Mbns as drug candidates for diseases involving dysregulation of copper homeostasis. Some of the most interesting work on Mbns is yet to come.

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